Proteins are biologically synthesized macromolecules having various roles in living systems. Enzymes are a particular variety of biologically active proteins which catalyze specific reactions. Presently, enzyme technology is used in many industrial applications and in basic research, such as, for example, medical research, food processing and preservation, the production of fermented beverages, the production of pharmaceuticals, and the analytical determination of the concentration of various metabolites in foods.
Enzymes are highly specific in their biological activity and generally catalyze a particular reaction at a very high rate compared to the corresponding reaction occurring at room temperature without biological catalysis. One enzyme may show catalytic activity with respect to a number of substrates upon which it can act. Accordingly, a given enzyme may catalyze the synthesis or degradation of more than one substrate.
Many enzymes are found in nature in very small quantities. Accordingly, their isolation, purification and use are limited to small-scale operations in view of the expense and time needed to isolate them in a usable amount.
Enzymes have been used extensively in industrial processes both in soluble and insoluble forms. The use of soluble enzymes for industrial processes, however, is limited by their cost, their instability, and the difficulties in recovering them after the operation.
These disadvantages have been circumvented by a new technology based on enzyme immobilization, i.e. enzyme attachment to solid support materials. A number of techniques have been developed for enzyme immobilization, of which the major ones are intra-molecular cross-linking and covalent linking to supports. Support materials in different forms--beads, membranes and fibers--can be used. The most common support material for enzyme reactors are beads or porous particles which can be packed into columns or used in stirred-tank reactors. Another form is the sheet or membrane form which can be used in pressure cells.
The advantage of utilizing such immobilized or insoluble enzymes resides in the possibility of acting catalytically with an enzyme on a substrate stream in a continuous way with no need of separating the enzyme from the product obtained by the catalytic reaction.
In short, many studies in biochemistry, biophysics, and chemical biology involve covalent cross-linking of enzymes and other proteins to different biomolecules and to carrier materials. All of the reagents developed to date for this purpose are bifunctional organic compounds. Some of them are selective, usually towards amino and sulfhydryl groups in the amino acid side chains; some, such as photogenerated nitrenes, are nonselective. Besides selectivity, desirable properties of cross-linking reagents are solubility in water; reactivity under mild, preferably physiological, conditions; stability; and cleavability, so that the linked species can be separated in their native forms.
All of these required properties can be achieved with inorganic reagents. Various spectroscopic and chemical properties render transition-metal complexes uniquely suited for specific covalent binding to amino-acid side chains in proteins and to other biological macromolecules.
An ideal cross-linking agent should be one which enhances the stability of the polymer under a wide variety of conditions. Also, an ideal cross-linking agent for proteins should be one which does not interfere with the activity of the protein, and in particular does not inactivate or denature the protein. Moreover, as earlier mentioned, ideal cross-linking agents should be soluble in water; they should also be active in neutral solutions, preferably in the physiological range of pH values.
Accordingly, a primary objective of the present invention is to provide stable cross-linked complexes between transition metal carboxylates and proteins.
A further objective of the invention is to provide a method of forming stable cross-link complexes between binuclear transition-metal compounds and proteins.
An even further objective of the present invention is to provide useful and improved cross-linked enzymes, with the cross-linking being between enzymatic proteins and dirhodium tetracarboxylates.
The method of accomplishing each of the above objectives of the present invention, as well as others, will become apparent from the detailed description of the invention which follows hereinafter.